Cell stressors, such as elevated levels of Reactive Oxygen Species (ROS), adversely affect Hemato... more Cell stressors, such as elevated levels of Reactive Oxygen Species (ROS), adversely affect Hematopoietic Stem Cell (HSC) reconstituting ability. However, the effects of ROS have not been evaluated in the context of hematopoietic development from human Pluripotent Stem Cells (hPSCs). Using our previously described in vitro system for efficient derivation of hematopoietic cells from hPSCs, we show that the vast majority of generated hematopoietic cells display supraphysiological levels of ROS compared to fresh cord blood cells. Elevated ROS resulted in DNA damage of the CD34 + hematopoietic fraction and, following functional assays, reduced colony formation and impaired proliferative capacity. Interestingly, all the proliferative potential of the most primitive hematopoietic cells was limited to a small fraction with low ROS levels. We show that elevation of ROS in hPSC-derived hematopoietic cells is contributed by multiple distinct cellular processes. Furthermore, by targeting these molecular processes with 4 unique factors, we could reduce ROS levels significantly, yielding a 22-fold increase in the most primitive CD90 + CD34 + hematopoietic cells with robust growth capacity. We demonstrate that the ROS reducing factors specifically reduced ROS in more primitive hematopoietic fractions, in contrast to endothelial cells that maintained low ROS levels in the cultures. We conclude that high levels of ROS in in vitro differentiation systems of hPSCs is a major determinant in the lack of ability to generate hematopoi-etic cells with similar proliferation/differentiation potential to in vivo hem-atopoietic progenitors, and suggest that elevated ROS is a significant barrier to generating hPSC-derived repopulating HSCs. STEM CELLS 2016; 00:000– 000 SIGNIFICANCE STATEMENT Cell stressors, such as elevated levels of Reactive Oxygen Species (ROS), adversely affect hematopoietic Stem Cell (HSC) reconstituting ability. However, the effects of ROS have not been evaluated in the context of hematopoietic development from human Pluripotent Stem Cells (hPSCs). Our results show for the first time that high levels of ROS, resulting from mechanisms associated with in vitro hPSC differentiation systems, occurs and functionally impairs the most primitive of the newly generated hematopoietic cells, and that ROS regulation is a requirement for generating functional primitive
SUMMARY Hematopoietic cells emerge from hemogenic endothelium in the developing embryo. Mechanism... more SUMMARY Hematopoietic cells emerge from hemogenic endothelium in the developing embryo. Mechanisms behind human hematopoietic stem and progenitor cell development remain unclear. Using a human pluripotent stem cell differentiation model, we report that cyclic AMP (cAMP) induction dramatically increases HSC-like cell frequencies. We show that hematopoietic cell generation requires cAMP signaling through the Exchange proteins activated by cAMP (cAMP-Epac) axis; Epac signaling inhibition decreased both hemogenic and non-hemogenic endothelium, and abrogated hematopoietic cell generation. Furthermore, in hematopoietic progenitor and stem-like cells, cAMP induction mitigated oxidative stress, created a redox-state balance, and enhanced C-X-C chemokine receptor type 4 (CXCR4) expression, benefiting the maintenance of these primitive cells. Collectively, our study provides insights and mechanistic details on the previously unrecognized role of cAMP signaling in regulating human hematopoietic development. These findings advance the mech-anistic understanding of hematopoietic development toward the development of transplantable human hematopoietic cells for therapeutic needs.
Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important f... more Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS. SAPs are also thought to be the cell of origin of neuroblastoma, a sympathetic PNS-related childhood tumor. However, it remains difficult to isolate sufficient numbers of SAPs. This protocol describes methodology for the generation of SAPs by differentiating murine embryonic stem cells (ESCs). These advances in SAP generation may facilitate investigations about the normal development and malignant transformation of the sympathetic PNS.
Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important f... more Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS.
The functions of retinoic acid (RA), a potent morphogen with crucial roles in embryogenesis inclu... more The functions of retinoic acid (RA), a potent morphogen with crucial roles in embryogenesis including developmental hematopoiesis, have not been thoroughly investigated in the human setting. Using an in vitro model of human hematopoietic development, we evaluated the effects of RA signaling on the development of blood and on generated hematopoietic progenitors. Decreased RA signaling increases the generation of cells with a hematopoietic stem cell (HSC)-like phenotype, capable of differentiation into myeloid and lymphoid lineages, through two separate mechanisms: by increasing the commitment of pluripotent stem cells toward the hematopoietic lineage during the developmental process and by decreasing the differentiation of generated blood progenitors. Our results demonstrate that controlled low-level RA signaling is a requirement in human blood development, and we propose a new interpretation of RA as a regulatory factor, where appropriate control of RA signaling enables increased ge...
Cell stressors, such as elevated levels of Reactive Oxygen Species (ROS), adversely affect Hemato... more Cell stressors, such as elevated levels of Reactive Oxygen Species (ROS), adversely affect Hematopoietic Stem Cell (HSC) reconstituting ability. However, the effects of ROS have not been evaluated in the context of hematopoietic development from human Pluripotent Stem Cells (hPSCs). Using our previously described in vitro system for efficient derivation of hematopoietic cells from hPSCs, we show that the vast majority of generated hematopoietic cells display supraphysiological levels of ROS compared to fresh cord blood cells. Elevated ROS resulted in DNA damage of the CD34 + hematopoietic fraction and, following functional assays, reduced colony formation and impaired proliferative capacity. Interestingly, all the proliferative potential of the most primitive hematopoietic cells was limited to a small fraction with low ROS levels. We show that elevation of ROS in hPSC-derived hematopoietic cells is contributed by multiple distinct cellular processes. Furthermore, by targeting these molecular processes with 4 unique factors, we could reduce ROS levels significantly, yielding a 22-fold increase in the most primitive CD90 + CD34 + hematopoietic cells with robust growth capacity. We demonstrate that the ROS reducing factors specifically reduced ROS in more primitive hematopoietic fractions, in contrast to endothelial cells that maintained low ROS levels in the cultures. We conclude that high levels of ROS in in vitro differentiation systems of hPSCs is a major determinant in the lack of ability to generate hematopoi-etic cells with similar proliferation/differentiation potential to in vivo hem-atopoietic progenitors, and suggest that elevated ROS is a significant barrier to generating hPSC-derived repopulating HSCs. STEM CELLS 2016; 00:000– 000 SIGNIFICANCE STATEMENT Cell stressors, such as elevated levels of Reactive Oxygen Species (ROS), adversely affect hematopoietic Stem Cell (HSC) reconstituting ability. However, the effects of ROS have not been evaluated in the context of hematopoietic development from human Pluripotent Stem Cells (hPSCs). Our results show for the first time that high levels of ROS, resulting from mechanisms associated with in vitro hPSC differentiation systems, occurs and functionally impairs the most primitive of the newly generated hematopoietic cells, and that ROS regulation is a requirement for generating functional primitive
SUMMARY Hematopoietic cells emerge from hemogenic endothelium in the developing embryo. Mechanism... more SUMMARY Hematopoietic cells emerge from hemogenic endothelium in the developing embryo. Mechanisms behind human hematopoietic stem and progenitor cell development remain unclear. Using a human pluripotent stem cell differentiation model, we report that cyclic AMP (cAMP) induction dramatically increases HSC-like cell frequencies. We show that hematopoietic cell generation requires cAMP signaling through the Exchange proteins activated by cAMP (cAMP-Epac) axis; Epac signaling inhibition decreased both hemogenic and non-hemogenic endothelium, and abrogated hematopoietic cell generation. Furthermore, in hematopoietic progenitor and stem-like cells, cAMP induction mitigated oxidative stress, created a redox-state balance, and enhanced C-X-C chemokine receptor type 4 (CXCR4) expression, benefiting the maintenance of these primitive cells. Collectively, our study provides insights and mechanistic details on the previously unrecognized role of cAMP signaling in regulating human hematopoietic development. These findings advance the mech-anistic understanding of hematopoietic development toward the development of transplantable human hematopoietic cells for therapeutic needs.
Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important f... more Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS. SAPs are also thought to be the cell of origin of neuroblastoma, a sympathetic PNS-related childhood tumor. However, it remains difficult to isolate sufficient numbers of SAPs. This protocol describes methodology for the generation of SAPs by differentiating murine embryonic stem cells (ESCs). These advances in SAP generation may facilitate investigations about the normal development and malignant transformation of the sympathetic PNS.
Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important f... more Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS.
The functions of retinoic acid (RA), a potent morphogen with crucial roles in embryogenesis inclu... more The functions of retinoic acid (RA), a potent morphogen with crucial roles in embryogenesis including developmental hematopoiesis, have not been thoroughly investigated in the human setting. Using an in vitro model of human hematopoietic development, we evaluated the effects of RA signaling on the development of blood and on generated hematopoietic progenitors. Decreased RA signaling increases the generation of cells with a hematopoietic stem cell (HSC)-like phenotype, capable of differentiation into myeloid and lymphoid lineages, through two separate mechanisms: by increasing the commitment of pluripotent stem cells toward the hematopoietic lineage during the developmental process and by decreasing the differentiation of generated blood progenitors. Our results demonstrate that controlled low-level RA signaling is a requirement in human blood development, and we propose a new interpretation of RA as a regulatory factor, where appropriate control of RA signaling enables increased ge...
Uploads